Siva K. Panguluri

Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice

Background Skeletal muscle wasting is a devastating complication of several physiological and pathophysiological conditions. Inflammatory cytokines play an important role in the loss of skeletal muscle mass in various chronic diseases. We have recently reported that proinflammatory cytokine TWEAK is a major muscle-wasting cytokine. Emerging evidence suggests that gene expression is regulated not only at transcriptional level but also at post-transcriptional level through the expression of specific non-coding microRNAs (miRs) which can affect the stability and/or translation of target mRNA. However, the role of miRs in skeletal muscle wasting is unknown. Methodology/Principal Findings To understand the mechanism of action of TWEAK in skeletal muscle, we performed mRNA and miRs expression profile of control and TWEAK-treated myotubes. TWEAK increased the expression of a number of genes involved in inflammatory response and fibrosis and reduced the expression of few cytoskeletal gene (e.g. Myh4, Ankrd2, and TCap) and metabolic enzymes (e.g. Pgam2). Low density miR array demonstrated that TWEAK inhibits the expression of several miRs including muscle-specific miR-1-1, miR-1-2, miR-133a, miR-133b and miR-206. The expression of a few miRs including miR-146a and miR-455 was found to be significantly increased in response to TWEAK treatment. Ingenuity pathway analysis showed that several genes affected by TWEAK are known/putative targets of miRs. Our cDNA microarray data are consistent with miRs profiling. The levels of specific mRNAs and miRs were also found to be similarly regulated in atrophying skeletal muscle of transgenic mice (Tg) mice expressing TWEAK. Conclusions/Significance Our results suggest that TWEAK affects the expression of several genes and microRNAs involved in inflammatory response, fibrosis, extracellular matrix remodeling, and proteolytic degradation which might be responsible for TWEAK-induced skeletal muscle loss.

RAPD and ISSR fingerprinting in cultivated chickpea (Cicer arietinum L.) and its wild progenitor Cicer reticulatum Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.

Tumor Necrosis Factor-α Regulates Distinct Molecular Pathways and Gene Networks in Cultured Skeletal Muscle Cells

Background Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood. Methodology/Principal Findings We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml) for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB) signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes. Conclusions TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-α in skeletal muscle cells.

PTTG: an important target gene for ovarian cancer therapy

Pituitary tumor transforming gene (PTTG), also known as securin is an important gene involved in many biological functions including inhibition of sister chromatid separation, DNA repair, organ development, and expression and secretion of angiogenic and metastatic factors. Proliferating cancer cells and most tumors express high levels of PTTG. Overexpression of PTTG in vitro induces cellular transformation and development of tumors in nude mice. The PTTG expression levels have been correlated with tumor progression, invasion, and metastasis. Recent studies show that down regulation of PTTG in tumor cell lines and tumors in vivo results in suppression of tumor growth, suggesting its important role in tumorigenesis. In this review, we focus on PTTG structure, sub-cellular distribution, cellular functions, and role in tumor progression with suggestions on possible exploration of this gene for cancer therapy.

AFLP fingerprinting in pigeonpea (Cajanus cajan (L.) Millsp.) and its wild relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.

MicroRNA signature of cis-platin resistant vs. cis-platin sensitive ovarian cancer cell lines

BackgroundOvarian cancer is the leading cause of death from gynecologic cancer in women worldwide. According to the National Cancer Institute, ovarian cancer has the highest mortality rate among all the reproductive cancers in women. Advanced stage diagnosis and chemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The most commonly employed chemotherapeutic drug for ovarian cancer treatment is cis-platin. As with most chemotherapeutic drugs, many patients eventually become resistant to cis-platin and therefore, diminishing its effect. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies.MethodsThe present study is focused on identifying the differential expression of regulatory microRNAs (miRNAs) between cis-platin sensitive (A2780), and cis-platin resistant (A2780/CP70) cell lines. Cell proliferation assays were conducted to test the sensitivity of the two cell lines to cis-platin. Differential expression patterns of miRNA between cis-platin sensitive and cis-platin resistant cell lines were analyzed using novel LNA technology.ResultsOur results revealed changes in expression of 11 miRNAs out of 1,500 miRNAs analyzed. Out of the 11 miRNAs identified, 5 were up-regulated in the A2780/CP70 cell line and 6 were down regulated as compared to cis-platin sensitive A2780 cells. Our microRNA data was further validated by quantitative real-time PCR for these selected miRNAs. Ingenuity Pathway Analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed for the selected miRNAs and their putative targets to identify the potential pathways and networks involved in cis-platin resistance.ConclusionsOur data clearly showed the differential expression of 11 miRNAs in cis-platin resistant cells, which could potentially target many important pathways including MAPK, TGF-β signaling, actin cytoskeleton, ubiquitin mediated proteasomal pathway, Wnt signaling, mTOR signaling, Notch signaling, apoptosis, and many other signaling pathways. Manipulation of one or more of these miRNAs could be an important approach for ovarian cancer chemotherapy.

MicroRNA-301a Mediated Regulation of Kv4.2 in Diabetes: Identification of Key Modulators

Diabetes is a metabolic disorder that ultimately results in major pathophysiological complications in the cardiovascular system. Diabetics are predisposed to higher incidences of sudden cardiac deaths (SCD). Several studies have associated diabetes as a major underlying risk for heart diseases and its complications. The diabetic heart undergoes remodeling to cope up with the underlying changes, however ultimately fails. In the present study we investigated the changes associated with a key ion channel and transcriptional factors in a diabetic heart model. In the mouse db/db model, we identified key transcriptional regulators and mediators that play important roles in the regulation of ion channel expression. Voltage-gated potassium channel (Kv4.2) is modulated in diabetes and is down regulated. We hypothesized that Kv4.2 expression is altered by potassium channel interacting protein-2 (KChIP2) which is regulated upstream by NFkB and miR-301a. We utilized qRT-PCR analysis and identified the genes that are affected in diabetes in a regional specific manner in the heart. At protein level we identified and validated differential expression of Kv4.2 and KChIP2 along with NFkB in both ventricles of diabetic hearts. In addition, we identified up-regulation of miR-301a in diabetic ventricles. We utilized loss and gain of function approaches to identify and validate the role of miR-301a in regulating Kv4.2. Based on in vivo and in vitro studies we conclude that miR-301a may be a central regulator for the expression of Kv4.2 in diabetes. This miR-301 mediated regulation of Kv4.2 is independent of NFkB and Irx5 and modulates Kv4.2 by direct binding on Kv4.2 3′untranslated region (3′-UTR). Therefore targeting miR-301a may offer new potential for developing therapeutic approaches.

Genetic Diversity of Iranian Accessions, Improved Lines of Chickpea (Cicer arietinum L.) and Their Wild Relatives by Using Simple Sequence Repeats

Biodiversity information in available germplasm is very useful for the success of any breeding program. To establish genetic diversity among 44 genotypes of chickpea comprising cultigen, landraces, internationally developed improved lines and wild relatives, genetic distances were evaluated using 19 simple sequence repeat markers with 100 marker loci. Estimation of the number of alleles per locus (na), the effective allele number (ne), and Wright fixation index F were 6.25, 3.67, and 0.44, respectively. Polymorphism information content values ranged from 0.84 (locus NCPGR6 and TA135) to 0.44 (locus NCPGR7) with an average of 0.68. Dice’s coefficient similarity matrix for studied chickpea genotypes varied from 0.07 to 1.0 indicating a broader genetic base among genotypes studied. The highest similarity, 1.0, was observed between genotypes Sel 96TH11484 and Sel 96TH11485; while, the lowest, 0.07, was observed between genotypes Sel 95TH1716 and Azad. Based on the UPGMA clustering method, all genotypes were clustered in eight groups, which indicated the probable origin and region similarity of landraces and local Iran landraces over the other cultivars and wild species. It also represents a wide diversity among available germplasm. Analysis of molecular variance revealed that 41% of the total variance was due to differences among groups while 59% was due to differences within groups. The results of principal coordinate analysis approximately corresponded to those obtained through cluster analysis. Genetic variation detected in this study can be useful for selective breeding for specific traits and in enhancing the genetic base of breeding programs.

Comparison of somatostatin and corticotrophin-releasing hormone immunoreactivity in forebrain neurons projecting to taste-responsive and non-responsive regions of the parabrachial nucleus in rat.

Several forebrain areas have been shown to project to the parabrachial nucleus (PBN) and exert inhibitory and excitatory influences on taste processing. The neurochemicals by which descending forebrain inputs modulate neural taste-evoked responses remain to be established. This study investigated the existence of somatostatin (SS) and corticotrophin-releasing factor (CRF) in forebrain neurons that project to caudal regions of the PBN responsive to chemical stimulation of the anterior tongue as well as more rostral unresponsive regions. Retrograde tracer was iontophoretically or pressure ejected from glass micropipettes, and 7 days later the animals were euthanized for subsequent immunohistochemical processing for co-localization of tracer with SS and CRF in tissue sections containing the lateral hypothalamus (LH), central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), and insular cortex (IC). In each forebrain site, robust labeling of cells with distinguishable nuclei and short processes was observed for SS and CRF. The results indicate that CRF neurons in each forebrain site send projections throughout the rostral caudal extent of the PBN with a greater percentage terminating in regions rostral to the anterior tongue-responsive area. For SS, the percentage of double-labeled neurons was more forebrain site specific in that only BNST and CeA exhibited significant numbers of double-labeled neurons. Few retrogradely labeled cells in LH co-expressed SS, while no double-labeled cells were observed in IC. Again, tracer injections into rostral PBN resulted in a greater percentage of double-labeled neurons in BNST and CeA compared to caudal injections. The present results suggest that some sources of descending forebrain input might utilize somatostatin and/or CRF to exert a broad influence on sensory information processing in the PBN.

Recent Patents on Antimicrobial Peptides

Research on antimicrobial peptides has gained pace to exploit their potential and ability to replace conventional antibiotics. Antimicrobial peptides are important members of the host defense system, as they have a broad ability to kill microbes. Antimicrobial peptides and proteins form an important means of host defense in eukaryotes. Large antimicrobial proteins (>100 a.a.), are often lytic, nutrient-binding proteins or specifically target the microbial macromolecules. Small antimicrobial peptides act by disrupting the structure or function of microbial cell membranes. A multitude of antimicrobial peptides has been found in the epithelial layers, phagocytes, and body fluids of multicellular animals including humans. Aside from their role as endogenous antibiotics, antimicrobial peptides have functions in inflammation, wound repair, and regulation of the adaptive immune system. In this review, we discuss recent patents relating to antimicrobial peptides. These patents are related to the method of identifying peptides that have antimicrobial activity, including the papillosin antimicrobial peptide and its encoding gene, the antimicrobial peptide isolated from Halocynthia aurantium, retrocyclins, and the use of cathelicidin LL-37 and its derivatives for wound healing. These patents provide valuable information that could be useful in the identification of antimicrobial peptides and the exploitation of their therapeutic potential.